TGF-beta Signaling and Degenerative Joint Diseases

描述（由申请人提供）：人骨关节炎（OA）是一种以关节软骨退化为特征的进行性关节疾病。骨性关节炎发展过程中通常发生的其他关键特征包括软骨下骨增厚、骨赘形成、滑膜不同程度的炎症、韧带和半月板退变以及关节囊肥大。然而，这些事件之间的后果和关系目前尚不清楚。tgf - β信号在控制软骨细胞分化中起关键作用。据报道，抑制tgf - β信号会导致小鼠出现严重的oa样表型。最近的遗传学研究也表明，Smad3基因突变与患者髋关节和膝关节OA的高发有关。然而，骨性关节炎发生过程中关节软骨细胞中TGFβ信号传导的关键下游靶基因仍有待确定。我们的初步研究表明，在出生后/成年期，软骨细胞特异性缺失II型TGFβ受体基因（Tgfbr2）会导致严重的oa样表型的发展。Tgfbr2条件敲除（Tgfbr2Col2ER）小鼠表现出OA最重要的特征。在这些小鼠中，Runx2和Atf4的表达显著增加。我们进一步证明，抑制TGFβ信号上调可调节Mmp13和Adamts5的表达。我们构建了Tgfbr2/Mmp13和Tgfbr2/Adamts5双敲除小鼠，并证明在Tgfbr2Col2ER背景中删除Mmp13或Adamts5基因显著逆转了Tgfbr2Col2ER小鼠中观察到的大多数OA特征。基于这些观察结果，我们假设Runx2与ATF4一起在激活Mmp13和Adamts5表达（Mmp13和Adamts5是软骨基质降解的两个关键酶）中发挥核心作用，从而导致Tgfbr2Col2ER小鼠的OA发展。在拟议的研究中，我们将利用综合的分子和遗传学方法确定TGFβ抑制->Runx2/ATF4->Mmp13/Adamts5通路在关节软骨细胞中的信号传导机制。我们提出了三个具体目标来检验我们的假设。在特异性目标1中：我们将确定TGFβ信号在成年小鼠OA发展中的作用。我们的假设是，成年小鼠关节软骨细胞中Tgfbr2基因的缺失导致成年Tgfbr2Col2ER小鼠的oa样形态学改变和疼痛相关行为改变。在特异性目标2中，我们将确定Mmp13在tgfbr2col2er诱导的OA表型中的作用。我们的假设是TGFβ信号抑制诱导的Mmp13表达是由转录因子Runx2和ATF4介导的。在特异性目标3中，我们将确定Adamts5在tgfbr2col2er诱导的OA表型中的作用。我们的假设是，在Tgfbr2Col2ER小鼠中，删除Adamts5/Mmp13基因比单独删除Mmp13和Adamts5对Tgfbr2Col2ER诱导的关节软骨降解有更好的保护作用。我们提出的研究将为OA发展的分子机制提供新的见解。

DESCRIPTION (provided by applicant): Human osteoarthritis (OA) is a progressive joint disease characterized by degradation of articular cartilage. Other key features normally occurring during OA development include thickening of the subchondral bone, formation of osteophyte, variable degrees of inflammation of the synovium, degeneration of ligaments and the menisci, and hypertrophy of the joint capsule. However, the consequence and relationship among these events are currently unknown. TGFβ signaling plays a critical role in controlling chondrocyte differentiation. It has been reported that inhibition of TGFβ signaling causes severe OA-like phenotype in mice. Recent genetic studies also demonstrated that mutations of the Smad3 gene are associated with high incidence of hip and knee OA in patients. However, the key downstream target genes of TGFβ signaling in articular chondrocytes during OA development remains to be determined. Our preliminary studies demonstrated that chondrocyte-specific deletion of the type II TGFβ receptor gene (Tgfbr2) at postnatal/adult stages lead to development of a severe OA-like phenotype. Tgfbr2 conditional knockout (Tgfbr2Col2ER) mice display the most important features of OA. In these mice Runx2 and Atf4 expression was significantly increased. We further demonstrated that inhibition of TGFβ signaling up regulates Mmp13 and Adamts5 expression. We generated Tgfbr2/Mmp13 and Tgfbr2/Adamts5 double knockout mice and demonstrated that deletion of either the Mmp13 or Adamts5 gene in the Tgfbr2Col2ER background significantly reversed the majority of the OA features observed in Tgfbr2Col2ER mice. Based on these observations, we hypothesize that Runx2, with ATF4, plays a central role in activation of Mmp13 and Adamts5 expression (MMP13 and ADAMTS5 are two key enzymes in cartilage matrix degradation) and consequently leading to OA development in Tgfbr2Col2ER mice. In the proposed studies we will determine the signaling mechanism of the TGFβ inhibition->Runx2/ATF4->Mmp13/Adamts5 pathway in articular chondrocytes using comprehensive molecular and genetic approaches. We have proposed three specific aims to test our hypothesis. In Specific Aim 1: we will determine the role of TGFβ signaling in OA development in adult mice. Our hypothesis is that deletion of the Tgfbr2 gene in articular chondrocytes of adult mice leads to OA-like morphological changes and pain-related behavioral changes in adult Tgfbr2Col2ER mice. In Specific Aim 2: we will determine the role of Mmp13 in Tgfbr2Col2ER-induced OA phenotype. Our hypothesis is that TGFβ signaling inhibition-induced Mmp13 expression is mediated by transcription factors Runx2 and ATF4. In Specific Aim 3: we will determine the role of Adamts5 in Tgfbr2Col2ER-induced OA phenotype. Our hypothesis is that deletion of Adamts5/Mmp13 genes in Tgfbr2Col2ER mice will have better protective effect than deletion of Mmp13 and Adamts5 alone on Tgfbr2Col2ER-induced articular cartilage degradation. Our proposed studies will provide novel insights into the molecular mechanisms of OA development.